Nonmuscle myosin 2A (NM 2A) not only plays numerous and diverse roles in vertebrate development, but also functions as a negative regulator for the formation of squamous cell carcinoma (SSC). Our previous research reported that ablation of NM 2A in mice (A-/A-) led to abnormal formation of the visceral endoderm and embryonic lethality by E7.5. However, the molecular mechanisms responsible for A-/A- embryonic lethality were not fully characterized. Since in vitro differentiation of embryonic stem cells (ESCs) has been shown to faithfully mimic in vivo early embryonic development, we first established mouse ESC differentiation systems, including a spontaneous differentiation system (embryonic body formation) and a hematopoietic differentiation system (embryonic body formation with enhanced hematopoietic differentiation). Using wild-type (A+/A+) and A-/A- ESCs as a model to test their pluripotency and differentiation potential, we found that the morphology of A-/A- ESCs was less organized and the surface cells were frequently scattered, in contrast to the tightly clustered A+/A+ ESCs. A-/A- ESCs significantly increased Sox2 and Nanog expression by almost 30 percent. Moreover, spontaneous differentiation of NM 2A deleted ESCs inhibited endoderm expression of genes, such as Sox17, Foxa2, and HNF4a. Analysis of the cell cycle with propidium iodide revealed that the peaks of G1 and G2/M shifted to the right in A-/A- embryonic body (EB) cells, indicating the increased binding of the propidium iodide to the DNA in the A-/A- cells compared to controls, by day 6 of EB formation. These data suggest that NM 2A deletion could affect chromatin structures. Using a nucleosomal DNA prep kit, it was determined that the genomic DNA of A-/A- ESCs or EBs was more easily digested into nucleosomes than control counterparts. Moreover, the levels of tri-methylation of K9 and K27 on histone H3 (H3K27me3 and H3K9me3, markers for repressive signals controlling development regulators), were significantly decreased in A-/A- EBs. These data indicate that there is a difference in chromatin structures between the wild-type and the A-/A- EBs. Because chromatin change has been demonstrated to be critical in early embryonic development, we hypothesize that ablation of NM 2A induces chromatin changes which consequently affect early embryonic development. Hematopoietic differentiation of ESCs in vitro is a useful method for investigating the molecular mechanisms controlling hematopoietic fate decisions. The ESC hematopoietic differentiation system has been widely used to study primitive hematopoiesis, including hematopoietic progenitors, primitive erythropoiesis, and primitive macrophage development. Flow cytometry analyses showed that A-/A- EBs yielded a 50 percent reduction in hematopoietic progenitors by day 6. Since primitive hematopoiesis occurs in mouse embryogenesis about E7.5, these data suggest that abnormal primitive hematopoiesis might be one cause of embryonic lethality of NM 2A knockout mice.